Salt of omecamtiv mecarbil and process for preparing salt

ABSTRACT

Provided are omecamtiv mecarbil dihydrochloride salt forms, compositions and pharmaceutical formulations thereof, and methods for their preparation and use.

FIELD

Provided are omecamtiv mecarbil dihydrochloride polymorph forms, methodsof making omecamtiv mecarbil, including omecamtiv mecarbildihydrochloride polymorph forms, compositions comprising omecamtivmecarbil dihydrochloride polymorph forms, and methods of using omecamtivmecarbil dihydrochloride salt polymorph forms.

BACKGROUND

The cardiac sarcomere is the basic unit of muscle contraction in theheart. The cardiac sarcomere is a highly ordered cytoskeletal structurecomposed of cardiac muscle myosin, actin and a set of regulatoryproteins. The discovery and development of small molecule cardiac musclemyosin activators would lead to promising treatments for acute andchronic heart failure. Cardiac muscle myosin is the cytoskeletal motorprotein in the cardiac muscle cell. It is directly responsible forconverting chemical energy into the mechanical force, resulting incardiac muscle contraction.

Current positive inotropic agents, such as beta-adrenergic receptoragonists or inhibitors of phosphodiesterase activity, increase theconcentration of intracellular calcium, thereby increasing cardiacsarcomere contractility. However, the increase in calcium levelsincrease the velocity of cardiac muscle contraction and shortenssystolic ejection time, which has been linked to potentiallylife-threatening side effects. In contrast, cardiac muscle myosinactivators work by a mechanism that directly stimulates the activity ofthe cardiac muscle myosin motor protein, without increasing theintracellular calcium concentration. They accelerate the rate-limitingstep of the myosin enzymatic cycle and shift it in favor of theforce-producing state. Rather than increasing the velocity of cardiaccontraction, this mechanism instead lengthens the systolic ejectiontime, which results in increased cardiac muscle contractility andcardiac output in a potentially more oxygen-efficient manner.

U.S. Pat. No. 7,507,735, herein incorporated by reference, discloses agenus of compounds, including omecamtiv mecarbil (AMG 423, CK-1827452),having the structure:

Omecamtiv mecarbil is a first in class direct activator of cardiacmyosin, the motor protein that causes cardiac contraction. It is beingevaluated as a potential treatment of heart failure in both intravenousand oral formulations with the goal of establishing a new continuum ofcare for patients in both the in-hospital and outpatient settings.

Because drug compounds having, for example, improved stability,solubility, shelf life, and in vivo pharmacology, are consistentlysought, there is an ongoing need for new or purer salts, hydrates,solvates, and polymorphic crystalline forms of existing drug molecules.The crystalline forms of omecamtiv mecarbil described herein help meetthis and other needs.

SUMMARY

Provided is a dihydrochloride form of omecamtiv mecarbil.

Also provided is omecamtiv mecarbil dihydrochloride hydrate.

Also provided is a crystalline form of a dihydrochloride form ofomecamtiv mecarbil.

Also provided is omecamtiv mecarbil dihydrochloride hydrate Form A.

Also provided is anhydrous omecamtiv mecarbil dihydrochloride.

Also provided is anhydrous omecamtiv mecarbil dihydrochloride Form B.

Also provided is anhydrous omecamtiv mecarbil dihydrochloride Form C.

Also provided are composition and pharmaceutical compositions comprisinga dihydrochloride form of omecamtiv mecarbil.

Also provided is a method of preparing omecamtiv mecarbil comprising

-   -   admixing methyl        4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate and phenyl        (6-methylpyridin-3-yl)carbamate in the presence of a        trialkylamine base to form omecamtiv mecarbil.

Also provided is a method of preparing omecamtiv mecarbildihydrochloride hydrate comprising:

-   -   (a) hydrogenating methyl        4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate in the        presence of a hydrogenation catalyst to form methyl        4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate;    -   (b) admixing methyl        4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate and phenyl        (6-methylpyridin-3-yl)carbamate in the presence of a        trialkylamine base to form omecamtiv mecarbil as a free base;        and    -   (c) crystallizing the omecamtiv mecarbil free base in the        presence of aqueous hydrochloric acid and an alcohol solvent to        form omecamtiv mecarbil dihydrochloride hydrate salt.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the dynamic vapor sorption of a omecamtiv mecarbildihydrochloride hydrate form, Form A.

FIG. 2 shows an X-ray powder diffraction pattern (XRPD) for Form A.

FIG. 3 shows an XRPD of a omecamtiv mecarbil dihydrochloride hydratesalt form at varying relative humidity conditions.

FIG. 4 shows an XRPD of a omecamtiv mecarbil dihydrochloride hydratesalt form at varying temperatures.

FIG. 5 shows the differential scanning calorimetry thermograph andthermogravimetric analysis for Form A.

FIG. 6 shows an overlay of XRPD patterns for Forms A, B and C ofomecamtiv mecarbil dihydrochloride salt.

FIG. 7A shows drug release at two pHs (2 and 6.8) for a formulation ofomecamtiv mecarbil free base hemihydrate.

FIG. 7B shows drug release of two pHs (2 and 6.8) for a omecamtivmecarbil dihydrochloride hydrate salt form, Form A.

DETAILED DESCRIPTION

Unless otherwise specified, the following definitions apply to termsfound in the specification and claims:

“Treatment” or “treating” means any treatment of a disease in a patient,including: a) preventing the disease, that is, causing the clinicalsymptoms of the disease not to develop; b) inhibiting the disease; c)slowing or arresting the development of clinical symptoms; and/or d)relieving the disease, that is, causing the regression of clinicalsymptoms. Treatment of diseases and disorders herein is intended to alsoinclude the prophylactic administration of a pharmaceutical formulationdescribed herein to a subject (i.e., an animal, preferably a mammal,most preferably a human) believed to be in need of preventativetreatment, such as, for example, chronic heart failure.

The term “therapeutically effective amount” means an amount effective,when administered to a human or non-human patient, to treat a disease,e.g., a therapeutically effective amount may be an amount sufficient totreat a disease or disorder responsive to myosin activation. Thetherapeutically effective amount may be ascertained experimentally, forexample by assaying blood concentration of the chemical entity, ortheoretically, by calculating bioavailability.

“Pharmaceutically acceptable salts” include, but are not limited tosalts with inorganic acids, such as hydrochlorate (i.e., hydrochloride),phosphate, diphosphate, hydrobromate, sulfate, sulfinate, nitrate, andlike salts; as well as salts with an organic acid, such as malate,maleate, fumarate, tartrate, succinate, citrate, acetate, lactate,methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate,salicylate, stearate, and alkanoate such as acetate, HOOC—(CH₂)_(n)—COOHwhere n is 0-4, and like salts. Similarly, pharmaceutically acceptablecations include, but are not limited to sodium, potassium, calcium,aluminum, lithium, and ammonium. Those skilled in the art will recognizevarious synthetic methodologies that may be used to prepare non-toxicpharmaceutically acceptable addition salts.

As used herein, the term “polymorphs” or “polymorphic forms” refers tocrystal forms of the same molecule. Different polymorphic forms of amolecule have different physical properties as a result of thearrangement or conformation of the molecules in the crystal lattice.Some of the different physical properties include melting temperature,heat of fusion, solubility, dissolution rate, and/or or vibrationalspectra. The physical form of a particular compound is particularlyimportant when the compound is used in a pharmaceutical formulationbecause different solid forms of a compound result in differentproperties of the drug product.

Polymorphs of a molecule can be obtained by a number of methods, asshown in the art, such as, for example, melt recrystallization, meltcooling, solvent recrystallization, desolvation, rapid evaporation,rapid cooling, slow cooling, vapor diffusion, and sublimation.Techniques for characterizing a polymorph include X-ray powderdiffraction (XRPD), single crystal X-ray diffraction (XRD), differentialscanning calorimetry (DSC), vibrational spectroscopy (e.g., IR and Ramspectroscopy), solid state nuclear magnetic resonance (ssNMR), hot stageoptical microscopy, scanning electron microscopy (SEM), electroncrystallography and quantitative analysis, particle size analysis (PSA),surface area analysis, solubility studies, and dissolution studies.

The term “hydrate” refers to the chemical entity formed by theinteraction of water and a compound.

As used herein, the term “monohydrate” refers a hydrate that containsone molecule of water per one molecule of the substrate.

As used herein, the term “crystalline” refers to a solid in which theconstituent atoms, molecules, or ions are arranged in a regularlyordered, repeating pattern in three dimensions.

The specification and claims contain listing of species using thelanguage “selected from . . . and . . . ” and “is . . . or . . . ”(sometimes referred to as Markush groups). When this language is used inthis application, unless otherwise stated it is meant to include thegroup as a whole, or any single members thereof, or any subgroupsthereof. The use of this language is merely for shorthand purposes andis not meant in any way to limit the removal of individual elements orsubgroups as needed.

Provided is a dihydrochloride hydrate form of omecamtiv mecarbil. Invarious embodiments of this aspect, the dihydrochloride hydrate form ofomecamtiv mecarbil is crystalline (Form A). Embodiments of thedihydrochloride hydrate form of omecamtiv mecarbil can be characterizedby one or more of the parameters described in further detail below.

The dihydrochloride hydrate form of omecamtiv mecarbil has a watersolubility of greater than 40 mg/mL at a pH in a range of about 3.5.Further, Form A is non-hygroscopic. For example, when subjected todynamic vapor sorption, Form A demonstrated a total weight gain of about0.55 wt. % between about 40% and about 95% relative humidity (RH) andweight loss of about 2.7 wt % between about 30% and about 5% RH. In someembodiments, the dihydrochloride hydrate form of omecamtiv mecarbil hasa dynamic vapor sorption profile substantially as shown in FIG. 1wherein by “substantially” is meant that the reported DVS features canvary by about ±5% RH.

The dynamic vapor sorption indicates that the salt dehydrates when driedto 5% relative humidity, but almost fully re-hydrates by 15% relativehumidity. Above 15% relative humidity, the sample is non-hygroscopic,showing only about a 1.0% weight change upon reaching 95% relativehumidity. No phase change occurred after the vapor sorption experimentwhen examined by XRPD.

Water solubility for Form A was determined to be greater than 40 mg/mL(pH=3.5) with no phase change occurring during the 24 hour slurryexperiment when examined by XRPD. Further still, Form A is stable underaccelerated stability testing conditions. For example, Form A remains insubstantially the same physical form over 6 months at 40° C. and 75% RH.

In various embodiments, Form A can be characterized by an X-ray powderdiffraction pattern, obtained as set forth in the Examples, having peaksat about 6.6, 14.9, 20.1, 21.4, and 26.8±0.2° 2θ using Cu Kα radiation.Form A optionally can be further characterized by an X-ray powderdiffraction pattern having additional peaks at about 8.4, 24.2, 26.0,33.3±0.2° 2θ using Cu Kα radiation. Form A optionally can be evenfurther characterized by an X-ray powder diffraction pattern havingadditional peaks at about 6.2, 9.7, 13.2, 14.3, 15.4, 16.3, 16.9, 18.9,19.5, 20.7, 21.8, 22.8, 23.6, 25.1, 27.3, 27.7, 28.4, 29.4, 30.2, 31.2,31.5, 31.9, 33.9, 34.5, 34.9, 36.1, 36.8, 37.7, 38.5, and 39.7±0.2° 2θusing Cu Kα radiation. In various cases, Form A can be characterized byan XRPD pattern having peaks at about 6.2, 6.6, 8.4, 9.7, 13.2, 14.3,14.9, 15.4, 16.3, 16.9, 18.9, 19.5, 20.1, 20.7, 21.4, 21.8, 22.8, 23.6,24.3, 25.1, 26.0, 26.8, 27.3, 27.7, 28.4, 29.4, 30.2, 31.2, 31.5, 31.9,33.3, 33.9, 34.5, 34.9, 36.1, 36.8, 37.7, 38.5, and 39.7±0.2° 2θ usingCu Kα radiation. In some embodiments, Form A has an X-ray powderdiffraction pattern substantially as shown in FIG. 2, wherein by“substantially” is meant that the reported peaks can vary by about±0.2°. It is well known in the field of XRPD that while relative peakheights in spectra are dependent on a number of factors, such as samplepreparation and instrument geometry, peak positions are relativelyinsensitive to experimental details.

Form B and Form C polymorphs of omecamtiv mecarbil, are metastableanhydrous dihydrochloride forms, and can be formed under variedhydration conditions, as noted in FIGS. 3, 4, and 6. Characteristic FormB 2-theta values include 6.8, 8.8, 14.7, 17.7, and 22.3±0.2° 2θ using CuKα radiation, and can additionally include peaks at 9.6, 13.5, 19.2,26.2±0.2° 2θ using Cu Kα radiation. Form B can be characterized withXRPD pattern peaks at 6.2, 6.8, 8.8, 9.6, 13.5, 14.4, 14.7, 15.4, 16.3,17.0, 17.7, 18.3, 19.2, 19.9, 20.5, 20.8, 21.8, 22.3, 22.7, 23.0, 24.8,25.1, 25.5, 26.2, 26.4, 26.8, 27.5, 28.5, 30.2, 30.6, 31.1, 31.5, 32.1,32.7, 34.1, 34.4, 35.5, 35.9, 38.1, 38.9±0.2° 2θ using Cu Kα radiation.Characteristic Form C 2-theta values include 6.7, 14.8, 17.4, 20.6, and26.2±0.2° 2θ using Cu Kα radiation, and can additionally include peaksat 8.7, 22.0, 27.1, and 27.7±0.2° 2θ using Cu Kα radiation. Form C canbe characterized with XRPD pattern peaks at 6.2, 6.7, 8.7, 9.6, 13.5,14.5, 14.8, 15.4, 16.4, 17.1, 17.4, 18.4, 19.3, 19.5, 19.9, 20.6, 20.8,21.8, 22.0, 22.5, 22.8, 24.3, 24.7, 25.1, 25.6, 26.2, 26.5, 27.1, 27.3,27.7, 28.5, 30.0, 30.5, 31.0, 31.5, 32.2, 32.8, 34.1, 35.2, 36.0, 36.9,and 38.8±0.2° 2θ using Cu Kα radiation. In some embodiments, Forms B andC have an X-ray powder diffraction pattern substantially as shown inFIG. 6, wherein by “substantially” is meant that the reported peaks canvary by about ±0.2°.

In various embodiments, Form A can be characterized by a single crystalx-ray diffraction (XRD) pattern, obtained as set forth in the Examplessection, wherein Form A comprises a triclinic space group of P-1 andunit cell parameters of about a=5.9979(4) {acute over (Å)}, b=13.4375(9){acute over (Å)}, c=14.4250(9) {acute over (Å)},

=97.617(4)°; β=93.285(4)°; and

=94.585(5)°. Form A optionally can be further characterized by the XRDparameters in the table, below.

Wavelength 1.54178 Å Crystal system Triclinic Space group P-1 Unit celldimensions a =5.9979(4) Å α =97.617(4) b =13.4375(9) Å β =93.285(4)° c=14.4250(9) Å

 =94.585(5)° Volume 1145.93(13) Å³ Z 2 Density (calculated) 1.427 Mg/m³Absorption 2.945 mm⁻¹ coefficient

DSC thermographs were obtained for Form A. The DSC curve indicates anendothermic transition that appears to be due to melting/decompositionaround 235° C. Thus, in embodiments, Form A can be characterized by aDSC thermograph having a decomposition endotherm with an onset in arange of about 230° C. to about 240° C. when Form A in an open aluminumpan. For example, in embodiments wherein Form A is heated from about 25°C. at a rate of about 10° C./min, Form A can be characterized by a DSCthermograph having a decomposition endotherm with an onset of about 235°C., as shown in FIG. 5.

Form A also can be characterized by thermogravimetric analysis (TGA).Thus, Form A can be characterized by a weight loss in a range of about2% to about 5% with an onset temperature in a range of about 100° C. toabout 150° C. For example, Form A can be characterized by a weight lossof about 3%, up to 150° C. In some embodiments, Form A has athermogravimetric analysis substantially as depicted in FIG. 5, whereinby “substantially” is meant that the reported TGA features can vary byabout ±5° C. This weight loss was determined to be water via KarlFischer (KF) analysis. KF analysis shows that the water content of FormA can be about 3.7, corresponding to a mono hydrate.

Form A can be characterized via variable temperature XRPD and variablerelative humidity XRPD. The variable temperature XRPD data are shown inFIG. 4. The data indicate that when Form A hydrate is heated beyond thedesolvation event shown in the TGA curve (about 75° C.), the materialconverts to a new dehydrated phase, Form B. When the material is cooledback down to ambient conditions, Form B resorbs water from theatmosphere and converts back to the hydrate Form A. The variablerelative humidity XRPD data are shown in FIG. 3. The data indicate thatwhen the hydrate Form A is exposed to 5% relative humidity, the materialconverts to a new dehydrated phase, Form C. When the material wasexposed to 15% relative humidity and higher, Form C resorbs water fromthe environment and converts back to the hydrate Form A. These data areconsistent with the vapor sorption experiment. An overlay of Form B andForm C are shown in FIG. 6. Arrows mark significant reflections of thetwo powder patterns indicating that the two phases are unique.

Also provided are compositions comprising a dihydrochloride hydrate formof omecamtiv mecarbil. In some embodiments, the compositions include atleast about 50, about 60, about 70, about 80, about 90, about 95, about96, about 97, about 98, or about 99% by weight of the dihydrochloridehydrate form of omecamtiv mecarbil. In some embodiments, thecompositions include at least about 50, about 60, about 70, about 80,about 90, about 95, about 96, about 97, about 98, or about 99% by weightof Form A of the dihydrochloride hydrate form of omecamtiv mecarbil. Insome embodiments, the compositions contain a mixture of two or more ofForms A, B, and C.

Also provided are pharmaceutical formulations comprising adihydrochloride hydrate form of omecamtiv mecarbil and at least onepharmaceutically acceptable excipient. In some embodiments, theformulations include at least about 50, about 60, about 70, about 80,about 90, about 95, about 96, about 97, about 98, or about 99% by weightof the dihydrochloride hydrate form of omecamtiv mecarbil. In someembodiments, the formulations include at least about 50, about 60, about70, about 80, about 90, about 95, about 96, about 97, about 98, or about99% by weight of Form A of the dihydrochloride hydrate form of omecamtivmecarbil. In some embodiments, the formulations contain a mixture of twoor more of Forms A, B, and C.

Also provided is a method for the use of such pharmaceuticalformulations for the treatment of heart failure, including but notlimited to: acute (or decompensated) congestive heart failure, andchronic congestive heart failure; particularly diseases associated withsystolic heart dysfunction.

Also provided is a synthesis of omecamtiv mecarbil comprising

-   -   admixing methyl        4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate and phenyl        (6-methylpyridin-3-yl)carbamate in the presence of a        trialkylamine base to form omecamtiv mecarbil.

In some embodiments, the weight ratio of phenyl(6-methylpyridin-3-yl)carbamate hydrochloride (i.e., SM-2 or phenylcarbamate) to methyl 4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate(i.e., SM-1 or piperazine nitro) is between about 1.1 and 1.5. In someembodiments, weight ratio of phenyl (6-methylpyridin-3-yl)carbamatehydrochloride to methyl4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate is about 1.2.

In some embodiments, the admixing is conducted in the presence of anaprotic solvent. In some embodiments, the solvent is THF.

In some embodiments, the trialkylamine base is triethylamine,diisopropylethylamine, or a combination thereof. In some embodiments,the trialkylamine base comprises diisopropylethylamine.

In some embodiments, an excess of the trialkylamine base is used. Insome embodiments, between about 1.1 and 1.5 equivalents of thetrialkylamine base is used. In some embodiments, about 1.3 equivalentsof the trialkylamine base is used.

In some embodiments, the admixing is conducted at 65° C.

In some embodiments, the method further comprises crystallizing theomecamtiv mecarbil in the presence of aqueous hydrochloric acid and analcohol solvent to form omecamtiv mecarbil dihydrochloride hydrate.

In some embodiments, the alcohol solvent comprises isopropyl alcohol.

In some embodiments, the aqueous hydrochloric acid comprises 6N HCl.

In some embodiments, the method further comprises mixing the omecamtivmecarbil dihydrochloride hydrate with at least pharmaceuticallyacceptable excipient to form a pharmaceutical formulation.

In some embodiments, the pharmaceutical formulation comprises omecamtivmecarbil dihydrochloride hydrate; a sweller layer; and a semi-permeablemembrane coating having at least one delivery port. The generalproperties of the drug layer and the sweller layer can be found in U.S.Pat. Pub. 2011/0182947, herein incorporated by reference.

In some embodiments, the pharmaceutical formulation is a modifiedrelease matrix tablet comprising omecamtiv mecarbil dihydrochloridehydrate; a control release agent; a pH modifying agent; a filler; and alubricant.

In some embodiments, the methyl4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate is prepared by aprocess comprising: hydrogenating methyl4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate in the presence of ahydrogenation catalyst to form methyl4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate.

In some embodiments, the hydrogenation catalyst comprises palladium. Insome embodiments, the hydrogenation catalyst is palladium on carbon.

Also provided is a method of preparing omecamtiv mecarbildihydrochloride hydrate comprising crystallizing omecamtiv mecarbil inthe presence of aqueous hydrochloric acid and an alcohol solvent to formomecamtiv mecarbil dihydrochloride hydrate.

In some embodiments, the alcohol solvent comprises isopropyl alcohol.

Also provided is a method of preparing omecamtiv mecarbildihydrochloride hydrate comprising:

-   -   (a) hydrogenating methyl        4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate in the        presence of a hydrogenation catalyst to form methyl        4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate;    -   (b) admixing methyl        4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate and phenyl        (6-methylpyridin-3-yl)carbamate in the presence of a        trialkylamine base to form omecamtiv mecarbil as a free base;        and    -   (c) crystallizing the omecamtiv mecarbil free base in the        presence of aqueous hydrochloric acid and an alcohol solvent to        form omecamtiv mecarbil dihydrochloride hydrate salt.

This synthesis provides high overall yields (greater than 70%). Inaddition, the dihydrochloride salt that results from the steps, can beformed as long rods when crystallized, having improved bulk properties,filtration times of minutes (compared to days for the free base form)and is highly soluble (greater than 40 mg/mL at pH 3.8). In variouscases, the resulting salt is the dihydrochloride hydrate Form A.

EXAMPLES General Methods

Reagents and solvents were used as received from commercial sources. ¹HNMR spectra were recorded on a 400 MHz spectrometer. Chemical shifts arereported in ppm from tetramethylsilane with the solvent resonance as theinternal standard (CDCl₃, DMSO-d₆). Data are reported as follows:chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, br=broad, m=multiplet), coupling constants (Hz) andintegration. ¹³C NMR spectra were recorded on a 100 MHz spectrometerwith complete proton decoupling. Chemical shifts are reported in ppmfrom tetramethylsilane with the solvent as the internal reference(CDCl₃, DMSO-d₆). All solvent charges are made with respect to starting2-Fluoro-3-nitrotoluene.

X-ray powder diffraction data was obtained using the Phillips x-rayautomated powder diffractometer (X'Pert) that was equipped with a fixedslit. The radiation was CuKα (1.541837 Å) and the voltage and currentwere 45 kV and 40 mA, respectively. Data was collected at roomtemperature from 3.000 to 40.009 degree 2-theta; step size was 0.008degrees; counting time was 15.240 seconds. Samples ranging from 5-40 mgwere prepared on the sample holder and the stage was rotated at arevolution time of 2.000 seconds.

The thermal properties of omecamtiv mecarbil bis-HCl salt werecharacterized using a DSC Q1000 or DSC Q 100 model, TA Instruments,differential scanning calorimetry, and a Q 500, TA Instruments,thermogravimetric analyzer. Data analysis was performed utilizingUniversal Analysis 2000, TA Instruments. Heating rates of 10 ° C./minwere used over a variety of temperature ranges for differential scanningcalorimetry and thermogravimetric analysis. Samples ranging from <1-5 mgwere prepared in crimped, hermetic or open aluminum pans for DSCanalysis.

Moisture balance data was collected using a VTI SGA 100 symmetricalvapor sorption analyzer. Relative humidity was varied in increments of5%, between 5% and 95% relative humidity during the adsorption run, andfrom 95% to 5% relative humidity during the desorption run. Equilibriumcriteria was set at 0.01% weight change in 1 minute with a maxequilibrium time of 180 minutes. Approximately 1-15 mg of sample wasused.

A colorless blade of C₂₀H₂₈C₁₂FN₅O₄, approximate dimensions 0.03 mm×0.12mm×0.50 mm, was used for the X-ray crystallographic analysis. The X-rayintensity data were measured at 100(2) K on a Bruker Kappa APEX IIsystem equipped with a graphite monochromator and a CuKα fine-focussealed tube (λ=1.54178 Å) operated at 1.2 kW power (40 kV, 30 mA). Thedetector was placed at a distance of 5.0 cm. from the crystal.

A total of 7824 frames were collected with a scan width of 0.5° in ω andφ and an exposure time of 90 sec/frame. The total data collection timewas 260 hours. The frames were integrated with the Bruker SAINT softwarepackage using a narrow-frame integration algorithm. The integration ofthe data using a Triclinic cell yielded a total of 12349 reflections toa maximum θ angle of 69.57° (0.83 Å resolution), of which 4046 wereindependent (redundancy 3.06), completeness=93.6%, R_(int)=5.13%,R_(sig)=5.18%) and 3351 (82.8%) were greater than >2 sigma(I) σ (F²).The final cell constants of a=5.9979(4)Å, b=13.4375(9) Å, c=14.4250(9)Å, α=97.617(4)°, β=93.285(4)°, γ=94.585(5)°, volume=1145.95(13)Å³, arebased upon the refinement of the XYZ-centroids of 4790 reflections above20σ(I) with 6.196°<2θ<138.239°. Analysis of the data showed negligibledecay during data collection. Data were corrected for absorption effectsusing the multiscan technique (SADABS). The ratio of minimum to maximumapparent transmission was 0.350. The calculated minimum and maximumtransmission coefficients (based on crystal size) are 0.3206 and 0.9168.

The structure was solved and refined using the Bruker SHELXTL (Version6.1) Software Package, using the space group P-1, with Z=2 for theformula unit, C₂₀H₂₈Cl₂FN₅O₄. The final anisotropic full-matrixleast-squares refinement on F² with 320 variables converged at R1=6.43%,for the observed data and wR2=19.18% for all data. The goodness-of-fitwas 1.067. The largest peak on the final difference electron densitysynthesis was 1.084 e⁻/Å³ and the largest hole was −0.527 e⁻/Å³ with anRMS deviation of 0.101 e⁻/Å³ On the basis of the final model, thecalculated density was 1.427 g/cm³ and F(000), 516 e⁻.

Two positions for partial water occupancies were found and refined inthis structure. The occupancies of the waters were refined independentlyto 53% and 41% for a total water content of 0.94 equivalents of waterper omecamtiv mecarbil molecule. This is consistent with other measuresof water content in this form of this compound. Hydrogen atoms on one ofthe solvating waters, the one with an occupancy of 41%, were found inthe electron density difference map and refined with bond lengths fixedat 1.01 Å. The hydrogen atoms on N3, C4 and N4 were found and allowed torefine isotropically. All other hydrogen atoms were placed at idealizedpositions and refined riding mode.

X-Ray powder diffraction data (XRPD) were obtained using a PANalyticalX'Pert PRO diffractometer (PANalytical, Almelo, The Netherlands) fittedwith a real time multiple strip (RTMS) detector. The radiation used wasCuKα(1.54 Å) and the voltage and current were set at 45 kV and 40 mA,respectively. Data were collected at room temperature from 5 to 45degrees 2-theta with a step size of 0.0334 degrees. Samples wereprepared on a low background sample holder and placed on the samplestage which was rotated with a 2 second revolution time.

Alternatively, XRPD data were obtained using a PANalytical X'Pert PROdiffractometer (PANalytical, Almelo, The Netherlands) fitted with a RTMSdetector. The radiation used was CuKα(1.54 Å) and the voltage andcurrent were set at 45 kV and 40 mA, respectively. Data were collectedat room temperature from 5 to 40, degrees 2-theta with a step size ofeither 0.0334 degrees. Samples were prepared on a low background sampleholder and placed on the sample stage which was rotated with a 2 secondrevolution time.

Alternatively, XRPD data were obtained using a PANalytical X'Pert PROdiffractometer (PANalytical, Almelo, The Netherlands) fitted with a RTMSdetector. The radiation used was CuKα(1.54 Å) and the voltage andcurrent were set at 45 kV and 40 mA, respectively. Data were collectedat room temperature from 5 to 40, degrees 2-theta with a step size ofeither 0.0167 degrees. Samples were prepared on a low background sampleholder and placed on the sample stage which was rotated with a 2 secondrevolution time.

Alternatively, XRPD data were obtained using a PANalytical X'Pert Prodiffractometer (PANalytical, Almelo, The Netherlands) fitted with a RTMSdetector. The radiation used was CuKα (1.54 Å) and the voltage andcurrent were set at 45 kV and 40 mA, respectively. Data were collectedat room temperature from 3 to 40, degrees 2-theta with a step size of0.008 degrees. Samples were prepared on a low background sample holderand placed on the sample stage with a 2 second revolution time.

Alternatively, XRPD data were obtained using a Bruker D8 Discover X-raydiffraction system (Bruker, Billerica, Mass.) fitted with a motorizedxyz sample stage and a GADDS area detector. The radiation used was CuKα(1.54 Å) and the voltage and current were set at 45 kV and 40 mA,respectively. The solid samples on a flat glass plate were mapped andfor each sample an area of 1 mm² was scanned in an oscillating mode for3 minutes from 5 to 48 degrees 2-theta.

Differential Scanning calorimetry (DSC) data was collected usingstandard DSC mode (DSC Q200, TA Instruments, New Castle, Del.). Aheating rate of 10° C./min was employed over a temperature range from40° C. to 300° C. Analysis was run under nitrogen and samples wereloaded in standard, hermetically-sealed aluminum pans. Indium was usedas a calibration standard.

Alternatively, DSC data were collected using temperature-modulated DSCmode (DSC Q200, TA Instruments, New Castle, Del.). After sampleequilibration at 20° C. for five minutes, the heating rate of 3° C./minwas employed with a modulation of +/−0.75° C./min over a temperaturerange from 20° C. to 200° C. Analysis was run under nitrogen and sampleswere loaded in standard, uncrimped aluminum pans. Indium was used as acalibration standard.

Manufacture of Omecamtiv Mecarbil Dihydrochloride Hydrate SyntheticRoute to Omecamtiv Mecarbil

Synthesis of the API SM Piperazine Nitro-HCl

In a 60 L reactor (containing no exposed Stainless steel, Hastelloy®, orother metal parts) equipped with a reflux/return condenser and scrubbercharged with a 5N NaOH solution, a mechanically stirred mixture ofFN-Toluene (2.0 kg, 12.89 mol, 1.0 equiv.), N-Bromosuccinimide (3.9 kg,21.92 mol, 1.70 equiv.), benzoyl peroxide (125.0 g, 0.03 equiv., 0.39mol, containing 25 wt % water), and acetic acid (7.0 L, 3.5 volumes) washeated to 85° C. under an atmosphere of nitrogen for 7 hours. A solutionof H₃PO₃ (106.0 g, 1.29 mol, 0.1 equiv.) and acetic acid (200 mL, 0.1volume), prepared in separate vessel, was added. The reaction mixturewas agitated for 0.5 h and analysis of an aliquot confirmed completedecomposition of benzoyl peroxide (not detected, HPLC_(254 nm)). Thereaction mixture was cooled to 22° C. DI Water (8.0 L, 4 volumes) andtoluene (16.0 L, 8 volumes) were charged, the biphasic mixture wasagitated (20 min), and the layers were separated. Aqueous 1.6N NaOH(14.0 L, 7.0 volumes) was added to the organic layer at a rate allowingthe batch temperature to stay under 25° C. and the pH of the resultantaqueous phase was measured (≥11). The biphasic mixture was filteredthrough a 5 μm Teflon® cartridge line and the layers were separated. Thefilter line was washed with another 2 L of toluene.

The assay yields were 2.5% of FN-Toluene, 62.3% of FN-Bromide and 30.0%of Di-Bromide. The toluene solution contained no benzoyl peroxide,succinimide, or α-bromoacetic acid and water content by KF titration was1030 ppm (This solution could be held under nitrogen at room temperaturefor >12 h without any change in the assay yield).

To this solution at room temperature was added diisopropylethylamine(880.0 g, 6.63 mol, 0.53 equiv.) followed by methanol (460 mL, 11.28mol, 0.88 equiv.) and heated to 40° C. A solution of diethylphosphite(820.0 g, 5.63 mol, 0.46 equiv.) in methanol (460 mL, 11.28 mol, 0.88equiv.) was prepared and added to the reaction mixture at 40° C. throughan addition funnel over a period of 1 hour at such a rate that the batchtemperature was within 40±5° C. The contents were stirred for a periodof 3 h at 40° C. from the start of addition and cooled to roomtemperature and held under nitrogen atmosphere for 12 hours. The assayyield of the reaction mixture was 2.5% FN-Toluene 92.0% FN-Bromide and0.2% Di-Bromide. This solution is used as such for the alkylation step.

Characterization for components of final product mixture (collected forpure compounds).

2-Fluoro-3-Nitrotoluene (FN-Toluene): ¹H NMR (400 MHz, CHLOROFORM-d) δppm 2.37 (s, 1 H), 7.13-7.20 (m, 1 H), 7.45-7.51 (m, 1 H), 7.79-7.85 (m,1 H). ¹³C NMR (100 MHz, CHLOROFORM-d) δ ppm 14.3 (d, J=5 Hz), 123.3 (d,J=3 Hz), 123.6 (d, J=5 Hz), 128.2 (d, J=16 Hz), 136.7 (d, J=5 Hz), 137.5(broad), 153.7 (d, J=261 Hz); 1-(bromomethyl)-2-fluoro-3-nitrobenzene(FN-Bromide): ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 4.56 (s, 1 H),7.28-7.34 (m, 1 H), 7.69-7.76 (m, 1 H), 7.98-8.05 (m, 1 H). ¹³C NMR (100MHz, CHLOROFORM-d) δ ppm 23.6 (d, J=5 Hz), 124.5 (d, J=5 Hz), 126.1 (d,J=3 Hz), 128.5 (d, J=14 Hz), 136.5 (d, J=4 Hz), 137.7 (broad), 153.3 (d,J=265 Hz). DSC: single melt at 53.59° C. Exact Mass [C₇H₅BrFNO₂+H]⁺:calc.=233.9566, measured=233.9561;1-(dibromomethyl)-2-fluoro-3-nitrobenzene (Dibromide): ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 6.97 (s, 1 H), 7.39-7.45 (m, 1 H), 8.03-8.10 (m, 1H), 8.16-8.21 (m, 1 H). ¹³C NMR (100 MHz, CHLOROFORM-d) δ ppm 29.2 (d,J=7 Hz), 124.9 (d, J=5 Hz), 127.1 (d, J=2 Hz), 132.1 (d, J=11 Hz), 135.7(d, J=2 Hz), 137.2 (broad), 149.8 (d, J=266 Hz). DSC: single melt at49.03° C. Exact Mass [C₇H₄Br₂FNO₂+H]⁺: calc.=311.8671,measured=311.8666.

Piperazine Nitro-HCl:

To a mechanically stirred toluene solution (9 volumes) of FN-Bromide(prepared from previous step) in a 60 L reactor at 22° C. under anatmosphere of nitrogen, diisopropylethylamine was charged (1.90 kg,14.69 mol, 1.14 equiv.). To this mixture a solution of piperazinecarboxylate methylester (Piperazine Carboxylate) (2.03 kg, 14.05 mol,1.09 equiv.) in toluene (1.0 L, 0.5 volumes) was added at a rateallowing the batch temperature to stay under 30.0° C. (Exothermic.During the addition, jacket temperature was adjusted to 5° C. in orderto maintain batch temperature below 30° C. The mixture was agitated at22° C. for 3 hours and analysis of an aliquot confirmed completion ofthe alkylation reaction (<1.0 LCAP FN-Bromide, HPLC_(254 nm)). Thereaction mixture was treated with aqueous NH₄Cl (20 wt %, 10.0 L, 5volumes; prepared from 2.0 kg of NH₄Cl and 10.0 L of DI water), thebiphasic mixture was agitated (30 min), and the layers were separated.The organic layer was sequentially washed with aqueous NaHCO₃ (9 wt %,10.0 L, 5 volumes; prepared from 0.90 kg of NaHCO₃ and 10.0 L of DIwater). The organic layer was filtered through a 5 μm Teflon® cartridgeline and transferred in a drum, washed the filter line with another 1.0L toluene and the combined toluene solution (10.0 volumes) weighed, andassayed (HPLC) to quantify Piperazine Nitro free base. The assay yieldfor the Piperazine Nitro-freebase is 89.0%, FN-Toluene 2.5% andFN-Bromide 0.2% with FN-Bromide undetected. The total loss of product tothe aqueous washes is <1.0%. This solution under nitrogen atmosphere isstable for more than 12 h.

To a mechanically stirred toluene solution of Piperazine Nitro freebase, prepared as described above, at 22° C. in a 60 L reactor under anatmosphere of nitrogen, IPA (19.4 L, 9.7 volumes) and DI water (1.0 L,0.5 volume) were charged. The mixture was heated to 55° C. and 20% ofthe 1.4 equiv. of conc. HCl (Titrated prior to use and charge based ontiter value; 276.0 mL, 3.21 mol) was charged. The contents were agitatedfor 15 min and Piperazine Nitro-HCl seed (130.0 g, 0.39 mol, 0.03equiv.) was charged as slurry in IPA (400 mL, 0.2 volume). The mixturewas agitated for 30 min and the remaining conc. HCl (80% of the charge,1.10 L, 12.82 mol) was added over a period of 4 hours. The mixture wasstirred at 55° C. for 1 h, cooled to 20° C. in a linear manner over 1.5hours, and agitated at this temperature for 12 hours. The supernatantconcentration of Piperazine Nitro-HCl was measured (2.8 mg/g). Themixture was filtered through an aurora filter equipped with a 5 μmTeflon® cloth. The mother liquor were transferred to a clean drum andassayed. The filter cake was washed twice with IPA (11.2 L, 5.6 volumes)and dried to constant weight (defined as ≤1.0% weight loss for 2consecutive TGA measurements over a period of 2 hours) on filter withvacuum and a nitrogen sweep (14 h). The combined losses of PiperazineNitro-HCl in the mother liquors and the washes were 2.5%. PiperazineNitro-HCl was isolated 3.59 kg in 87.6% corrected yield with >99.5 wt %and 99.0% LCAP purity.

Methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate hydrochloride(Piperazine Nitro-HCl): ¹H NMR (300 MHz, DMSO-d) δ ppm 3.25 (br. s, 3H), 3.52-3.66 (m, 8 H), 4.47 (s, 2 H), 7.44-7.63 (t, 1 H, J=8 Hz),7.98-8.15 (m, 1 H), 8.17-8.34 (m, 1 H). ¹³C NMR (75 MHz, DMSO-d) δ ppm50.3, 51.4, 52.8, 119.6 (d, J=14 Hz), 125.1 (d, J=5 Hz), 127.9, 137.4(d, J=8 Hz), 139.8 (d, J=3 Hz), 152.2, 154.7, 155.7. DSC: melt onset at248.4° C. Exact Mass [C₁₃H₁₆FN₃O₄+H]⁺: calculated=298.1203,measured=298.1198.

Alternative Processes for the Synthesis of Piperazine Nitro:

A mixture of NaBH₄ (1.7 g, 44 mmol) in THF (68 mL) was treated2-fluoro-3-nitrobenzoic acid (3.4 g, 18.4 mmol) and cooled to 0-5° C. Asolution of iodine (4.7 g, 18.4 mmol) in THF (12 mL) was then added dropwise at a rate to control off-gassing. The progress of the reaction wasassessed by HPLC. After 2 hours HPLC assay indicated 4% AUC of2-fluoro-3-nitrobenzoic acid remained. The mixture was quenched into 1 MHCl (30 mL) and extracted with MTBE (5 mL). The organics were thenwashed with 20% aqueous KOH solution and 10% sodium thiosulfate. Theorganics were dried with Na₂SO₄, filtered over Celite and concentratedto afford (2-fluoro-3-nitrophenyl)methanol (2.8 g, 88%, 89% AUC byHPLC).

A solution of (2-fluoro-3-nitrophenyl)methanol (2.8 g, 16 mmol) in2-MeTHF (26 mL) was treated with triethylamine (4.5 mL, 32 mmol) andcooled to 0-5° C. The solution was then treated with methanesulfonylchloride (1.6 mL, 21 mmol). The progress of the reaction was assessed byHPLC. After 30 minutes at 0-5° C., the reaction was deemed complete. Themixture was quenched with water (14 mL) and the phases were separated.The organics were washed with brine, dried with Na₂SO₄, filtered overCelite and concentrated to afford 2-fluoro-3-nitrobenzylmethanesulfonate (3.3 g, 83.1%, 81% AUC by HPLC) as a yellow oil.

A solution of 2-fluoro-3-nitrobenzyl methanesulfonate (3.3 g, 13 mmol,AMRI lot # 46DAT067B) in toluene (33 mL), was treated withdiisopropylethylamine (2.7 mL, 15 mmol) in one portion. A solution ofmethylpiperazine-1-carboxylate (2.1 g, 15 mmol) in toluene (1.1 mL) wasadded slowly via syringe to maintain between 23-29° C. The reaction wasstirred for 16 hours following the addition. An HPLC assay after thistime showed that the reaction was complete. 20% Aqueous NH₄Cl (11 mL)was added at 20-25° C. The biphasic mixture was stirred for 15 minutes,and the phases were separated. This process was repeated using 9%aqueous sodium bicarbonate (11 mL). The toluene layer was then filteredover Celite at 20-25° C. 2-propanol (50 mL) and water (1.1 mL) wereadded to the toluene solution and the mixture heated to 55-60° C. Themixture was then treated with 37 wt % HCl (1.6 mL, 18.7 mmol) over 20minutes. A precipitate was noted following the addition. When theaddition was complete, the mixture was allowed to cool gradually to20-25° C. and was stirred for hours before filtering and washing withIPA (2 bed volumes).

The cake was then dried at under vacuum to afford4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate hydrochloride (2.41g, 54%, 90% AUC by HPLC, 88 wt % by HPLC).

Piperazine Nitro Freebase:

In a 60 L reactor equipped with a reflux/return condenser, a mixture ofPiperazine Nitro-HCl (2.0 kg, 5.99 mol, 1.0 equiv.) and isopropylacetate (6.0 L, 3.0 volumes) was mechanically agitated at ambienttemperature under an atmosphere of nitrogen. A solution of sodiumbicarbonate (629 g, 7.49 mol, 1.25 equiv.) and water (7.5 L, 3.75volume), prepared in separate vessel, was added. The biphasic mixturewas agitated (15 min), and the layers were separated. The upper organiclayer (containing product) was transferred to a separate vessel whilethe reactor was rinsed with water and isopropanol. The organic layer wasthen transferred through an inline 5 μm Teflon® cartridge back into theclean 60 L reactor. The filter line was washed with 4.0 L (2.0 volumes)of isopropanol into the 60 L reactor. An additional 12.0 L (6.0 volumes)of isoproponal was added to the 60 L reactor and heated to 40° C. Underreduced pressure (50 torr) the batch was concentrated down toapproximately 6 L (3.0 volumes). The solution was cooled from 27° C. to20° C. in a linear manner over 10 minutes. Water (4.0 L, 2.0 volumes)was added at 20° C. over 30 minutes followed by Piperazine NitroFreebase seed (18 g, 0.06 mol, 0.01 equiv). The mixture was aged for 5minutes and the remaining water (24.0 L, 12.0 volumes) was added over 90minutes. After holding overnight at 20° C., the supernatantconcentration of Piperazine Nitro Freebase was measured (<10 mg/mL). Themixture was filtered through an aurora filter equipped with a 12 μmTeflon® cloth. The filter cake was washed with a mixture of water (3.3L, 1.65 volumes) and isopropanol (700 mL, 0.35 volumes) and dried toconstant weight (defined as ≤1.0% weight loss for 2 consecutive TGAmeasurements over a period of 2 hours) on filter with vacuum and anitrogen sweep (48 h). The combined losses of Piperazine Nitro Freebasein the mother liquors and the wash were aproximately 7.5%. PiperazineNitro Freebase was isolated 1.67 kg in 92.5% corrected yield with 100.0wt % and 99.4% LCAP purity.

Synthesis of the API SM Phenyl Carbamate-HCl

A 60 L, glass-lined, jacketed reactor set at 20° C. under nitrogenatmosphere and vented through a scrubber (containing 5N NaOH) wascharged with 2.5 kg of Amino Pyridine (1.0 equiv, 23.1 moles), followedby 25 L (19.6 kg, 10 vol) acetonitrile. After initiating agitation and(the endothermic) dissolution of the Amino Pyridine, the vessel wascharged with 12.5 L of N-methyl-2-pyrolidinone (12.8 kg, 5 vol). Anaddition funnel was charged with 1.8 L (0.6 equiv, 13.9 moles) phenylchloroformate which was then added over 68 minutes to the solution ofthe Amino Pyridine keeping the internal temperature≤30° C. The reactionwas agitated for >30 minutes at an internal temperature of 20±5° C. Thevessel was then charged with 61±1 g of seed as a slurry in 200 mLacetonitrile and aged for ≥30 min. The addition funnel was charged with1.25 L (0.45 equiv, 9.7 moles) of phenyl chloroformate which was thenadded over 53 minutes to the reaction suspension while again keeping thetemperature≤30° C. The contents of the reactor were aged≥30 hours at20±5° C. After assaying the supernatant (≤15 mg/g for both product andstarting material), the solids were filtered using an Aurora filterequipped with a 12 μm Teflon cloth. The mother liquor was forwarded to a2^(nd) 60 L, glass-lined, jacketed reactor. The reactor and cake wererinsed with 1×10 L of 5:10 NMP/ACN and 1×10 L ACN. The washes wereforwarded to the 2^(nd) reactor as well. The cake was dried under vacuumwith a nitrogen bleed for 24 hours to afford 5.65 kg (90.2% yield) ofthe product, Phenyl Carbamate-HCl as an off-white solid in 98.8 wt %with 99.2% LCAP purity.

Phenyl (6-methylpyridin-3-yl)carbamate hydrochloride (PhenylCarbamate-HCl) ¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.24 (s, 1 H), 8.81 (s,1 H), 8.41 (d, 1 H, J=8.8 Hz), 7.85 (d, 1 H, J=8.8 Hz), 7.48-7.44 (m, 2H), 7.32-7.26 (m, 3 H), 2.69 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ ppm151.66, 150.01, 147.51, 136.14, 133.79, 129.99, 129.49, 127.75, 125.87,121.70, 18.55: HR-MS: Calclulated for C₁₃H₁₂N₂O₂: 228.0899,M+H⁺=229.0972; Observed mass: 229.0961

Alternative Synthesis of Phenyl Carbamate HCl

5-Amino-2-methylpyridine (53.2 kg, 1.0 equiv) and acetonitrile (334 kg,8.0 mL/g) were charged to a nitrogen flushed glass-lined reactor. Thecontents of the reactor were stirred while warming to 25-30° C. Themixture was then recirculated through a filter packed with activatedcarbon (11 kg, 20 wt %) for 3 h intervals while maintaining 25-30° C.Following each 3 h interval, a sample of the mixture was analyzed forcolor by comparison to a color standard and UV Absorbance at 440 nm.Once a satisfactory result was achieved, the filter was blown out intothe reactor and the filter was rinsed with acetonitrile (85 kg, 2.0mL/g). The acetonitrile rinse was transferred into the reaction mixture.1-Methyl-2-pyrrolidinone (274 kg, 5.0 mL/g) was charged to the reactionmixture in the glass-lined reactor. Phenyl chloroformate (46.6 kg, 0.6equiv) was slowly added to the mixture while maintaining 15-30° C.(typically 60-70 min). The reaction mixture was stirred forapproximately 60 minutes while maintaining 20-25° C.Phenyl(6-methylpyridin-3-yl)carbamate hydrochloride (0.58 kg, 0.010equiv) seed crystals were charged to the stirring mixture. The slurrywas then stirred for approximatly 4 h at 20±5° C. Phenyl chloroformate(33.4 kg, 0.45 equiv) was slowly added to the slurry while maintaining15-30° C. The mixture was then allowed to age while stirring for 8±1 hwhereupon concentration of 5-amino-2-methylpyridine (target≤15 mg/mL)and phenyl (6-methylpyridin-3-yl)carbamate hydrochloride (target≤15mg/mL) were checked by HPLC. The batch was then filtered under vacuumand washed with a mixture of acetonitrile (112 kg, 2.68 mL/g) and1-methyl-2-pyrrolidinone (72 kg, 1.32 mL/g) followed by washing thrisewith acetonitrile (167 kg, 4.0 mL/g). The solids were deliquoredfollowed by transfering to a tray dryer maintained between 20-40° C. and1.3-0.65 psia until an LOD of <1 wt % was achieved, whereuponphenyl(6-methylpyridin-3-yl)carbamate hydrochloride 106.3 kg (81.6%yield) was isolated from the dryer.

Methyl 4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate (PiperazineAniline)

To a 100-L jacketed glass-lined reactor were added methyl4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate hydrochloride (2.00kg, 1.00 equiv) and isopropyl acetate (6.00 L, 3.00 Vol with-respect tostarting material). The resulting slurry was agitated under a nitrogensweep. To the mixture was added dropwise over 45±30 min: 7.7% w/waqueous sodium bicarbonate solution (629 g, 1.25 equiv of sodiumbicarbonate dissolved in 7.50 L water), maintaining an internaltemperature of 20±5° C. by jacket control (NOTE: addition isendothermic, and may evolve up to 1 equiv of carbon dioxide gas). Themixture was stirred for ≥15 min, resulting in a clear biphasic mixture.Agitation was stopped and the layers were allowed to settle.

The bottom (aqueous) layer was drained and analyzed by pH paper toensure that the layer is pH>6. Quantititative HPLC analysis of the upper(organic) layer revealed 97-100% assay yield of the methyl4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate freebase (1.73-1.78kg). The upper (organic) layer was transferred through an in-line filterinto a 20-L Hastelloy® hydrogenator, and the 100-L reactor and lineswere rinsed with an additional aliquot of isopropyl acetate (2.00 L,1.00 Vol). The hydrogenator was purged with nitrogen and vented toatmospheric pressure. To the reaction mixture was added a slurry of 5.0wt % palladium on carbon (20.0 g, Strem/BASF Escat™ 1421, approx 50%water) in isopropyl acetate (400 mL), followed by a 400 mL rinse. Theresulting reaction mixture was diluted with an additional aliquot ofisopropyl acetate (1.2 L; total isopropyl acetate amount is 10.0 L, 5.00Vol). The hydrogenator was purged three times with nitrogen (pressurizedto 60±10 psig, then vented to atmospheric pressure), then pressurized to60±5 psig with hydrogen. The reaction mixture was stirred at <100 rpm at30±5° C. while maintaining 60±5 psig hydrogen, for >2 hours untilreaction was deemed complete. This temperature and pressure correspondto a measured kLa value of approx 0.40 in a 20-L Hydrogenator. End ofreaction is determined by dramatic decrease in hydrogen consumptionaccompanied by a relief in the heat evolution of the reaction. Tocontrol potential dimeric impurities, the reaction is continued for atleast 30 minutes after this change in reaction profile, and HPLCanalysis is performed to confirm that >99.5% conversion of thehydroxyl-amine to the aniline is achieved.

At the end of reaction, the hydrogenator was purged with nitrogen twice(pressurized to 60±10 psig, then vented to atmospheric pressure). Thecrude reaction mixture was filtered through a 5 μm filter followed by a0.45 μm filter in series, into a 40-L glass-lined reactor. Thehydrogenator and lines were washed with an additional aliquot ofisopropyl acetate (2.00 L). Quantitative HPLC analysis of the crudereaction mixture revealed 95-100% assay yield (1.52-1.60 kg anilineproduct). The reaction mixture was distilled under reduced pressure(typically 250-300 mbar) at a batch temperature of 50±5° C. until thetotal reaction volume was approximately 8.00 L (4.00 Vol). The batch wassubjected to a constant-volume distillation at 50±5° C., 250-300 mbar,by adding heptane to control the total batch volume. After approximately8.00 L (4.00 Vol) of heptane were added, GC analysis indicated that thesolvent composition was approximately 50% isopropyl acetate, 50%heptane. Vacuum was broken, and the internal batch temperature wasmaintained at 50±5° C. To the reaction mixture was added a slurry ofseed (20.0 grams of product methyl4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate, in a solvent mixtureof 80 mL heptane and 20 mL isopropyl acetate). The resulting slurry wasallowed to stir at 50±5° C. for 2±1 hours, then cooled to 20±5° C. over2.5±1.0 h. Additional heptane (24.0 L, 12.0 Vol) was added dropwise over2 hours, and the batch was allowed to stir at 20±5° C. for ≥1 hours(typically overnight). Quantitative HPLC analysis of this filteredsupernatant revealed <5 mg/mL product in solution, and the productcrystals were 50-400 μm birefringent rods. The reaction slurry wasfiltered at 20° C. onto a filter cloth, and the cake wasdisplacement-washed with heptane (6.00 L, 2.00 Vol). The cake was driedon the filter under nitrogen sweep at ambient temperature for >4 hours,until sample dryness was confirmed by LOD analysis (indicated <1.0 wt %loss). The product methyl4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate (1.56 kg) wasisolated as a pale-yellow powder in 86% yield at 99.8 wt % by HPLC with100.0 LCAP₂₁₀. [Analysis of the combined filtrates and washes revealed108 grams (7.0%) of product lost to the mother liquors. The remainingmass balance is comprised of product hold-up in the reactor (fouling).]¹H NMR (DMSO-d₆, 400 MHz) δ: 6.81 (dd, J=7.53, 7.82 Hz, 1H), 6.67 (m,1H), 6.49 (m, 1H), 5.04 (s, 2H), 3.58 (s, 3H), 3.45 (m, 2H), 3.34 (m,4H), 2.33 (m, 4H). ¹⁹F NMR (d₆-DMSO, 376 MHz) δ: −140.2. ¹³C NMR(d₆-DMSO, 125 MHz) δ: 155.0, 150.5, 148.2, 136.2 (m), 123.7 (m), 117.6,115.1, 73.7, 54.9 (m), 52.1 (m), 43.4. mp=89.2° C.

Alternate Route to Piperazine Aniline

To a jacketed glass-lined reactor were added methyl4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate hydrochloride (46.00kg, 1.00 equiv) and isopropyl acetate (200 kg, 5.0 mL/g). The resultingslurry was agitated under a nitrogen sweep. To the mixture was added7.4% w/w aqueous sodium bicarbonate solution (1.25 equiv) whilemaintaining an internal temperature of 25±5° C. The mixture was agitatedfor ≥30 min, resulting in a clear biphasic mixture. Agitation wasstopped and the bottom (aqueous) layer was discharged. Analysis ofaqueous layer indicates pH≥6. Water (92 kg, 2.0 mL/g) was charged theorganic layer and agitated for ≥15 min. Agitation was then stopped andthe bottom (water wash) layer was discharged. Water (92 kg, 2.0 mL/g)was charged the organic layer and agitated for ≥15 min. Agitation wasthen stopped and the bottom (water wash) layer was discharged. The batchwas distilled under reduced pressure while maintaining the batchtemperature between 40-50° C. The batch volume was held constantthroughout the distillation by the continuous addition of isopropylacetate. Once the water content of the batch was <1,500 ppm, thesolution was passed through an inline filter into a Hastelloy reactorcontaining 5.0 wt % palladium on carbon (BASF Escat 1421, 0.69 kg, 1.5wt %). The jacketed glass-lined reactor was rinsed with isopropylacetate (100 kg, 2.5 mL/g) and added to the Hastelloy reactor though theinline filter.

The batch was adjusted to approximately 25-35° C. (preferably 30° C.)and hydrogen gas was added to maintain about 4 barg with vigorousagitation. Hydrogenation was continued for 1 h after hydrogen uptake hasceased, and ≥99.0% conversion by HPLC were achieved. The palladium oncarbon catalyst was collected by filtration and the supernatant wascollected in a reactor. Isopropyl acetate (40 kg, 1.0 mL/g) was chargedto the Hastelloy reactor and transferred through the filter andcollected in the jacketed glass-lined reactor.

The batch was concentrated under reduced pressure while maintaining thebatch temperature between 35-55° C. until the final volume wasapproximately 4.0 mL/g. Heptane (219 kg, 7.0 mL/g) was added to thejacketed glass-lined reactor while maintaining the batch between 50-60°C., until 20-25% isopropyl acetate in heptane was achieved as measuredby GC. The solution was cooled to between 40-50° C. and seeded withmethyl 4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate (0.46 kg, 1.0wt %) as a slurry in heptane (6.4 kg, 0.20 mL/g). The slurry was agedfor approximately 2 h, whereupon, the batch was distilled under reducedpressure while maintaining the batch temperature between 35-45° C. Thebatch volume was held constant throughout the distillation by thecontinuous addition of heptane (219 kg, 7.0 mL/g). The batch was thencooled to between 15-25° C. over approximately 3 h. Concentration of thesupernatant was measured to be ≤5 mg/mL methyl4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate by HPLC.

The batch was filtered and the resulting solids were successively washedwith heptane (63 kg, 2.0 mL/g) then heptane (94 kg, 3.0 mL/g). Thesolids were dried on the filter with a stream of dry nitrogen withvacuum until an LOD of ≤1 wt % was achieved whereupon 33.88 kg (90.7%yield) was isolated from the filter dryer.

Omecamtiv Mecarbil Dihydrochloride Hydrate Procedure

To a 15 L glass lined reactor were charged methyl4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate (1,202 g, 4.50 mol),phenyl (6-methylpyridin-3-yl)carbamate hydrochloride (1,444 g, 5.40mol), and tetrahydrofuran (4.81 L). The resulting slurry was agitatedunder a nitrogen sweep and N,N-diisopropylethylamine (1,019 L, 5.85 mol)was then charged to the slurry which resulted in a brown solution. Thetemperature of the solution was increased to 65° C. and agitated for 22h, until <1% AUC piperazine aniline remained by HPLC analysis.

The batch was cooled to 50° C. and distilled under reduced pressurewhile maintaining the internal temperature of the vessel below 50° C. byadjusting vacuum pressure. 2-Propanol was added with residual vacuum ata rate to maintain a constant volume in the 15 L reactor. A total of10.5 kg of 2-propanol was required to achieve <5% THF by GC. Water (2.77kg) was then charged to the reactor followed by the addition of 6N HCl(1.98 kg) at a rate to maintain the internal temperature below 60° C.The reactor was brought to ambient pressure under a nitrogen sweep. Thesolution was then heated to 60° C., and transferred to a 60 L glasslined reactor through an inline filter. The 15 L reactor was then rinsedwith 1:1 water/2-propanol (1.2L) which was sent through the inlinefilter to the 60 L reactor.

The 60 L reactor was adjusted to 45° C. and a slurry of seed (114 g,0.23 mol) in 2-propanol (0.35 L) was added to the reactor resulting in aslurry. The batch was aged at 45° C. for 1 h, followed by the additionof 2-propanol (3.97 kg) through an inline filter over 2 h. The batch washeated to 55° C. over 1 h and held for 0.25 h, then cooled back to 45°C. over 1 h and held overnight at 45° C. 2-propanol (11.71 kg) was thenadded through an inline filter to the batch over 3 h. The batch was agedfor 1 h and then cooled to 20° C. over 2 h and held at 20° C. for 0.5 h.The batch was then recirculated though a wet mill affixed with 1-mediumand 2-fine rotor-stators operating at 56 Hz for 2.15 h, until no furtherparticle size reduction was observed by microscopy.

The batch was then filtered through a 20″ Hastelloy® filter fitted witha 12 um filter cloth under 500 torr vacuum. A wash solution of 95:52-propanol:water (1.82 L) was charged through an inline filter to the 60L reactor, then onto the filter. A second wash of 2-propanol (2.85 L)was charged through an inline filter to the 60 L reactor, then onto thefilter. The batch was then dried under 5 psi humidified nitrogenpressure until <5,000 ppm 2-propanol, and 2.5-5% water remained. Thefinal solid was discharged from the filter to afford 2.09 kg of methyl4-(2-fluoro-3-(3-(6-methylpyridin-3-yl)ureido)benzyl)piperazine-1-carboxylateas an off-white crystalline solid in 89% yield at 99.88 wt % by HPLC,100.0% AUC. Total losses to liquors was 0.10 kg (4.7%).

DSC: T_(onset)=61.7° C., T_(max)=95.0° C.; TGA=2.2%, degradationonset=222° C.; ¹H HMR (D₂O, 500 MHz) δ 8.87 (s, 1H), 8.18 (d, J=8.9 Hz,1H), 7.83 (t, J=7.5 Hz, 1H), 7.71 (d, J=8.8 Hz, 1H), 7.35-7.29 (m, 2H),4.48 (s, 2H), 4.24 (br s, 2H), 3.73 (s, 3H), 3.31 (br s, 6H), 2.68 (s,3H); ¹³C HMR (D₂O, 150 MHz) δ 156.8, 154.2, 153.9 (J=249 Hz), 147.8,136.3, 136.1, 130.1, 129.4, 128.0, 127.2, 125.5 (J=11.8 Hz), 125.1(J=4.2 Hz), 116.1 (J=13.5 Hz), 53.54, 53.52, 53.49, 50.9, 40.5, 18.2.

Alternative Process for the Coupling (Aniline Phenyl Carbamate)

A reaction vessel was charged methyl4-(3-amino-2-fluorobenzyl)piperazine-1-carboxylate (2.5 g, 1.0 equiv),acetonitrile (25.0 mL, 10.0 mL/g) and 1-methyl-2-pyrrolidinone (12.5 mL,5.0 mL/g). The batch was cooled to 0° C. whereupon phenyl chloroformate(1.20 mL, 1.02 equiv) was added over approximately 5 min. After 45minutes the resulting slurry resulted was allowed to warm to 20° C. Thesolids were collected by filtration and rinsed twice with acetonitrile(10.0 mL, 4.0 mL/g). The solids were dried under a stream of drynitrogen to afford methyl4-(2-fluoro-3-((phenoxycarbonyl)amino)benzyl)piperazine-1-carboxylatehydrochloride 2.8 g (71% yield) as a white solid.

4-(2-fluoro-3-((phenoxycarbonyl)amino)benzyl)piperazine-1-carboxylatehydrochloride: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.08 (br. s., 2 H),3.24-3.52 (m, 4 H), 3.62 (s, 3 H), 4.03 (d, J=11.25 Hz, 2 H), 4.38 (br.s., 2 H), 7.11-7.35 (m, 4 H), 7.35-7.49 (m, 2 H), 7.49-7.66 (m, 1 H),7.80 (s, 1 H), 10.12 (br. s, 1 H), 11.79 (br. s, 1 H); HRMS=388.1676found, 388.1667 calculated.

A reaction vessel was charged methyl4-(2-fluoro-3-((phenoxycarbonyl)amino)benzyl)piperazine-1-carboxylatehydrochloride (0.50 g, 1.0 equiv), 6-methylpyridin-3-amine (0.15 g, 1.2equiv), tetrahydrofuran (2.0 mL, 4.0 mL/g) and N,N-diisopropylethylamine(0.23 mL, 1.1 equiv). The batch was heated to 65° C. for 22 h, whereuponquantitative HPLC analysis indicated 0.438 g (92% assay yield) ofomecamtiv mecarbil.

Alternative Omecamtiv Mecarbil Dihydrochloride Hydrate Procedure

Omecamtiv Mecarbil, free base (3.0 kg, 1.0 equiv) was charged to anitrogen purged jacketed vessel followed by water (4.6 L, 1.5 mL/g) and2-propanol (6.1 L, 2.60 mL/g). The slurry was agitated and heated toapproximately 40° C., whereupon 6N HCl (2.6 L, 2.10 equiv) was chargedto the slurry resulting in a colorless homogenous solution. The solutionwas heated to between 60-65° C. and transferred through an inline filterto a 60 L reactor pre-heated to 60° C. The batch was cooled to 45° C.whereupon Omecamtiv Mecarbil dihydrochloride hydrate (150 g, 5.0 wt %)was charged to the vessel as a slurry in 95:5 (v/v) 2-Propanol/Water(600 mL, 0.20 mL/g). The resulting slurry was maintained at 45° C. for0.5 h followed by cooling to approximately 20° C. then held for 3-16 h.2-Propanol (33.0 L, 11.0 mL/g) was added over ≥2 h followed by a ≥1 hisothermal hold at approximately 20° C. (Supernatant pH≤7).

The batch was recirculated through a wet mill for 5-10 batch turnoversuntil sufficient particle reduction was achieve as compared to offlinecalibrated visual microscopy reference. The slurry was filtered byvacuum and the resulting solids were washed with two washes of 95:5(v/v) 2-Propanol/Water (3.0 L, 1.0 mL/g) and a final cake wash with2-Propanol (6.0 L, 2.0 mL/g). The cake was dried on the filter bypushing humidified nitrogen through the cake until ≤5,000 ppm 2-propanoland 2.5-5% water were measured by GC and KF analysis, respectively.Omecamtiv Mecarbil dihydrochloride hydrate was isolated as a colorlesscrystalline solid (3.40 kg, 93% yield).

pH Dependent Release Profiles

A formulation of omecamtiv mecarbil hemihydrate (free base) anddihydrochloride hydrate (Form A) were prepared having the followingcomponents, all components reported as a w/w %:

Free Base (75 mg matrix tablet) Active granulation: 15.37% free base;30% hypromellose, HPMC K100 MPrem CR; 10% citric acid monohydrate;11.88% microcrystalline cellulose, Avicel PH 101; 6.75% lactosemonohydrate, FastFlo 316; 12.5% purified water; and CitricAcid granulation: 20% citric acid monohydrate; 5% microcrystallinecellulose, Avicel PH 101; and 1% magnesium stearate, non-bovine.Form A (75 mg matrix tablet) Intra-granulation: 18.37% Form A; 30%hypromellose, HPMC K100 MPrem CR; 0.50% magnesium stearate;; andExtra-granulation: 16.88% microcrystalline cellulose, Avicel PH 101;18.37% citric acid anhydrous; and 0.5% magnesium stearate, non-bovine.

The formulations were tested at pH 2 and pH 6.8 and the amount of drugreleased over time was measured. The results of this drug releaseprofile are shown in FIG. 6.

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosed salts or polymorphs.Variations and changes which are obvious to one skilled in the art areintended to be within the scope and nature of the invention which aredefined in the appended claims.

What is claimed:
 1. A dihydrochloride salt of omecamtiv mecarbil.
 2. Theform of claim 1, wherein the dihydrochloride salt is a monohydrate. 3.The salt of claim 1 or 2, wherein the salt is crystalline.
 4. The saltof any one of claims 1 to 3, wherein the salt is characterized by anX-ray powder diffraction pattern comprising peaks at about 6.6, 14.9,20.1, 21.4, and 26.8±0.2° 2θ using Cu Kα radiation.
 5. The salt of claim4, further comprising peaks at about 8.4, 24.2, 26.0, and 33.3±0.2° 2θusing Cu Kα radiation.
 6. The salt of claim 4 or 5, further comprisingpeaks at about 6.2, 9.7, 13.2, 14.3, 15.4, 16.3, 16.9, 18.9, 19.5, 20.7,21.8, 22.8, 23.6, 25.1, 27.3, 27.7, 28.4, 29.4, 30.2, 31.2, 31.5, 31.9,33.9, 34.5, 34.9, 36.1, 36.8, 37.7, 38.5, and 39.7±0.2° 2θ using Cu Kαradiation.
 7. The salt of any one of claims 1 to 6, having an X-raypowder diffraction pattern substantially as shown in FIG.
 8. 8. The saltof any one of claims 1 to 7, having a endothermic transition at about230° C. to about 240° C., as measured by differential scanningcalorimetry.
 9. The salt of claim 8, wherein the transition is at about235° C.
 10. A method of preparing omecamtiv mecarbil dihydrochloridehydrate comprising: (a) hydrogenating methyl4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate in the presence of ahydrogenation catalyst to form methyl4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate; (b) admixing methyl4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate and phenyl(6-methylpyridin-3-yl)carbamate in the presence of a trialkylamine baseto form omecamtiv mecarbil as a free base; and (c) crystallizing theomecamtiv mecarbil free base in the presence of aqueous hydrochloricacid and an alcohol solvent to form omecamtiv mecarbil dihydrochloridehydrate salt.
 11. The method of claim 10, further comprising formulatingomecamtiv mecarbil dihydrochloride hydrate salt.
 12. The method of claim10, wherein the hydrogenation catalyst comprises palladium.
 13. Themethod of claim 12, wherein the hydrogenation catalyst is palladium oncarbon.
 14. The method of any one of claims 10 to 13, wherein thetrialkylamine base is triethylamine, diisopropylethylamine, or acombination thereof.
 15. The method of any one of claims 10 to 14,wherein the trialkylamine base comprises diisopropylethylamine.
 16. Themethod of any one of claims 10 to 15, wherein the alcohol solventcomprises isopropyl alcohol.
 17. The method of any one of claims 10 to16, wherein the omecamtiv mecarbil dihydrochloride hydrate salt has anx-ray powder diffraction pattern (XRPD) comprising peaks at about 6.6,14.9, 20.1, 21.4, and 26.8±0.2° 2θ using Cu Kα radiation.
 18. The methodof claim 17, wherein the XRPD pattern further comprises peaks at about8.4, 24.2, 26.0, and 33.3±0.2° 2θ using Cu Kα radiation.
 19. The methodof claim 17 or 18, wherein the XRPD pattern further comprises peaks atabout 6.2, 9.7, 13.2, 14.3, 15.4, 16.3, 16.9, 18.9, 19.5, 20.7, 21.8,22.8, 23.6, 25.1, 27.3, 27.7, 28.4, 29.4, 30.2, 31.2, 31.5, 31.9, 33.9,34.5, 34.9, 36.1, 36.8, 37.7, 38.5, and 39.7±0.2° 2θ using Cu Kαradiation.
 20. A method of preparing omecamtiv mecarbil comprisingadmixing methyl 4-(3-amino-2-fluorobenzyl)piperazine-1-caboxylate andphenyl (6-methylpyridin-3-yl)carbamate in the presence of atrialkylamine base to form omecamtiv mecarbil.
 21. The method of claim20, wherein the trialkylamine base comprises diisopropylethylamine. 22.The method of claim 20 or 21, further comprising crystallizing theomecamtiv mecarbil in the presence of aqueous hydrochloric acid and analcohol solvent to form an omecamtiv mecarbil dihydrochloride hydratesalt.
 23. The method of claim 22, wherein the alcohol solvent comprisesisopropyl alcohol.
 24. The method of claim 22 or 23, wherein theomecamtiv mecarbil dihydrochloride hydrate salt has an x-ray powderdiffraction pattern (XRPD) comprising peaks at about 6.6, 14.9, 20.1,21.4, and 26.8±0.2° 2θ using Cu Kα radiation.
 25. The method of claim24, wherein the XRPD pattern further comprises peaks at about 8.4, 24.2,26.0, and 33.3±0.2° 2θ using Cu Kα radiation.
 26. The method of claim 24or 25, wherein the XRPD pattern further comprises peaks at about 6.2,9.7, 13.2, 14.3, 15.4, 16.3, 16.9, 18.9, 19.5, 20.7, 21.8, 22.8, 23.6,25.1, 27.3, 27.7, 28.4, 29.4, 30.2, 31.2, 31.5, 31.9, 33.9, 34.5, 34.9,36.1, 36.8, 37.7, 38.5, and 39.7±0.2° 2θ using Cu Kα radiation.